This application claims priority from Canadian application no. 2,246,005, filed on Oct. 1, 1998, which is incorporated by reference in its entirety.
The invention relates to hybrid nucleic acid molecules and vectors for expression, at single copy, of RNA, polypeptides and for gene therapy in erythroid and other cells. In particular, the invention relates to hybrid nucleic acid molecules and vectors that are useful for treatment of hemoglobinopathies such as sickle cell anemia and xcex2- or xcex1-thalassemia.
The invention also relates to hybrid nucleic acid molecules that are useful for erythroid expression at single copy of RNA and polypeptides in transgenic animals.
One difficult aspect of gene therapy is in reproducibly obtaining high-level, tissue-specific, and long-term expression from nucleic acid molecules transferred into stem cells [1]. Since commonly used retrovirus and Adeno-Associated Virus (AAV) vectors may integrate at single copy, their transduced nucleic acid molecules should be regulated by tissue-specific elements that function at single copy. Locus Control Regions (LCR) are well suited for this task as they direct reproducible expression from all integration sites and transgene copy numbers[2], indicating that they have transcriptional enhancement and chromatin opening activities. For example, the human xcex2-globin LCR directs high level xcex2-globin transgene expression in erythroid cells of transgenic mice regardless of the integration site. However, it has become apparent that the xcex2-globin LCR cannot confer reproducible transgene expression in mice on other nucleic acid molecule sequences such as the LacZ marker gene [3, 4], xcex3-globin genes [5-7], or even xcex2-globin genes that lack a 3xe2x80x2 element [8, 9]. These findings suggest that chromatin opening by the LCR requires xcex2-globin gene sequences, and that the utility of this LCR is limited to expression of the xcex2-globin gene. It is not clear what gene sequences would be useful. In contrast to expression in transgenic mice, chromatin opening activities are not required for transient expression [4] or for stable expression when under selection for a drug resistance gene [10]. Therefore, such in vitro assays are not well suited for the evaluation of minigene cassettes for use in retrovirus-mediated gene therapy.
Efficient cell-specific nucleic acid molecule expression at some but not all transgene integration sites can be achieved by using cell-specific gene proximal elements including promoter elements, cell-specific regulatory elements such as enhancers and silencers, RNA processing signals, and cell-specific RNA-stabilizing elements. Cell-specific gene expression at all transgene integration sites primarily results from locus control regions (LCRs). However, it is considered to be very difficult to design a hybrid nucleic acid molecule that expresses at all integration sites for gene therapy of erythroid cells or their precursors because there is inadequate information about regulation of erythroid gene expression by LCRs and of the requirement by LCRs for specific gene proximal elements. Currently, no suitable hybrid nucleic acid molecule that expresses at single copy at all transgene integration sites for erythroid or precursor cell gene therapy has been reported.
The invention is a hybrid nucleic acid molecule for expressing RNA and polypeptides specifically in cells of the erythroid lineage. It is particularly useful for gene therapy of diseases such as thalassemias and sickle cell anemia by delivery of therapeutically useful globin proteins in erythroid cells. Other RNA and polypeptides may also be delivered with the hybrid nucleic acid molecules for gene therapy or in transgenic animals.
The invention uses human xcex2-globin DNA regulatory elements to obtain high level, reproducible, tissue specific, single copy expression of coding nucleic acid molecules. Unlike past attempts to use xcex2-globin DNA regulatory elements in preparing vectors, the invention uses much smaller portions of xcex2-globin DNA regulatory elements. The simultaneous presence of the xcex2-globin promoter, intron 2 and 3xe2x80x2 enhancer and the 5xe2x80x2HS3 is sufficient to provide coding nucleic acid molecule expression, for example the xcex3-globin gene. The hybrid nucleic acid molecule is suitable for DNA or viral mediated gene therapy of sickle cell anemias and thalassemias.
The invention also includes an isolated nucleic acid molecule comprising xcex2-globin DNA regulatory elements that is capable of having a coding nucleic acid molecule inserted in it. The molecule is capable of directing production of a polypeptide at single copy in a targeted cell of the erythroid lineage.
In a preferred embodiment, the invention relates to hybrid nucleic acid molecules for gene therapy in erythroid and other cells, and in particular xcex1-, xcex2- xcex4-, xcex5-, xcex3-, or xcex6-globin nucleotide sequences, or derivatives thereof, operably linked to xcex2-globin regulatory elements. The hybrid nucleic acid molecules are useful for treatment of hemoglobinopathies such as sickle cell anemia or xcex2- or xcex1-thalassemia.
The invention also includes a hybrid nucleic acid molecule for producing a protein in a targeted cell, preferably an erythroid cell, consisting of:
xcex2-globin DNA regulatory elements, and
a nucleic acid molecule operatively associated with the regulatory elements and capable of expression in the cell.
Other hemoglobinopathy or erythroid diseases, disorders or abnormal physical states that require erythroid specific expression may also be treated. They are useful for expressing any genes in cells of the erythroid lineage in mammals, such as mice or human, such as genes encoding Glucose 6 Phosphate Dehydrogenase or ferrochelatase polypeptides. Marker genes may also be used, such as green fluorescent protein.
In a preferred embodiment, the hybrid nucleic acid molecule consists of all or part of the nucleotide sequence of the BGT50 construct shown in FIG. 8. The xcex2-globin DNA regulatory elements are preferably human xcex2-globin DNA regulatory elements. The regulatory elements are preferably a 5xe2x80x2HS3, a promoter, a 3xe2x80x2 enhancer and intron 2.
The invention also includes a composition, preferably a pharmaceutical composition, including a therapeutically effective amount of the hybrid nucleic acid molecule and a pharmaceutically acceptable carrier. Another embodiment of the invention relates to a composition comprising the hybrid nucleic acid molecule and a carrier.
In another embodiment, the invention relates to the use of a hybrid nucleic acid molecule for treatment of a disease, disorder or abnormal physical state, including hemoglobinopathy. Another embodiment of the invention includes a method of medical treatment of hemoglobinopathy comprising administering a hybrid nucleic acid molecule to a patient and expressing the nucleic acid molecule.
The invention also includes a method of designing a hybrid nucleic acid molecule for treatment of a hemoglobinopathy. The method involves generating a series of hybrid nucleic acid molecules including erythroid cell-specific regulatory elements and then assessing each hybrid nucleic acid molecule single copy transgenic mice for the expression of reporter nucleic acid molecules or the hybrid nucleic acid molecule.
The invention includes a hybrid nucleic acid molecule for treating a defect in a globin gene in a cell of an erythroid lineage, the hybrid nucleic acid molecule including: a) xcex2-globin gene regulatory elements which elements are capable of regulating gene expression in the cell; and b) a coding nucleic acid molecule operatively associated with the regulatory elements and capable of expression in the cell, the coding nucleic acid molecule encoding a globin protein or a derivative thereof having globin activity. The defect being treated causes a hemoglobinopathy. The target cell may be an erythroid cell. The invention also includes a cell of an erythroid lineage containing recombinant xcex2-globin DNA regulatory elements and a coding nucleic acid molecule operatively associated with the regulatory elements, the cell expressing polypeptides, such as globins, not normally expressed by the cell at biologically significant levels.
Another aspect of the invention includes DNA sequences which are complementary to the aforementioned sequences.
Another embodiment of the invention relates to a hybrid nucleic acid molecule that at single copy is capable of producing a RNA and a polypeptide in a targeted mammalian cell of the erythroid lineage, including:
xcex2-globin DNA regulatory elements, and
a coding nucleic acid molecule operatively associated with the regulatory elements and capable of expression in the cell.
The hybrid nucleic acid molecule CAN INCLUDE all or part of a nucleic acid molecule selected from th group consisting of
a) SEQ ID NO:1 or SEQ ID NO:2, or a complement thereof
b) SEQ ID NO:3 or SEQ ID NO:4, or a complement thereof
c) a nucleic acid molecule that hybridizes to all or part of a nucleic acid molecule shown in SEQ ID NO:1-SEQ ID NO:4, or a complement thereof under high stringency hybridization conditions; and
d) a nucleic acid molecule having at least 70% identity with the nucleotide sequence of (a) or (b).
The mammal is preferably a human. The xcex2-globin DNA regulatory elements may include 5xe2x80x2HS3, a xcex2-globin promoter, a xcex2-globin intron 2 and a xcex2-globin 3xe2x80x2 enhancer. In another embodiment the xcex2-globin DNA regulatory elements may include an 5xe2x80x2HS3, Axcex3-globin promoter, a xcex2-globin intron 2 and a xcex2-globin 3xe2x80x2 enhancer. The coding sequence preferably encodes a globin polypeptide or a derivative thereof having globin activity, such as xcex1-, xcex2-, xcex4-, xcex5-, xcex3- or xcex6-globin. The xcex2-globin DNA regulatory elements may include human xcex2-globin DNA regulatory elements. The regulatory elements may include a 5xe2x80x2HS3, a promoter, a 3xe2x80x2 enhancer and intron 2.
The hybrid nucleic acid molecule preferably includes 5xe2x80x2-A-X-Y-C-D-3xe2x80x2 wherein A is a 5xe2x80x2HS3, X includes a xcex2-globin promoter or an Axcex3-globin promoter, Y includes a coding nucleic acid molecule, C includes a xcex2-globin intron 2, and D includes a xcex2-globin 3xe2x80x2 enhancer. The coding nucleic acid molecule preferably encodes a globin polypeptide or a derivative thereof having globin activity.
The invention also includes a vector including a nucleic acid molecule of the invention. The invention also includes a composition including a vector or nucleic acid molecule of the invention. Another aspect of the invention is a host cell including a vector or nucleic acid molecule of the invention. The host cell can include a cell of the erythroid lineage selected from the group including an erythroid cell, an erythroid cell precursor, a bone marrow cell, an umbilical cord blood cell, a hematopoietic stem cell, a hematopoietic stem cell that is CD34xe2x88x92/CD38xe2x88x92, or CD34+/CD38xe2x88x92, a progenitor cell of the erythroid lineage, CFU-GEMM, BFU-E, CFU-E, a pro-erythroblast, an erythroblast and an erythrocyte.